Plasmacytoid dendritic cells (pDCs) are critical innate immune cells that secrete high levels of type I interferons (IFNs) in response to a variety of viruses. Type I IFN system plays a pivotal role in the early innate immune responses against virus infection. One of the major pathways of pathogen detection involves the recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) found on microbes via pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs). Recognition of pathogens via TLRs induces activation of DCs and the initiation of the adaptive immune responses necessary for clearance of and protection from a given pathogen. Viruses are recognized by both cytosolic sensors and endosomal sensors. The pDCs almost exclusively utilize the endosomal TLRs for viral recognition. In previous studies we demonstrated the requirement of TLR9 and TLR7 in the activation and IFN1 secretion in response to double stranded (ds) DNA viruses, herpes simplex viruses (HSV), and single stranded (ss) RNA viruses, influenza and vesicular stomatitis virus (VSV), respectively. The recognition of both types of viruses through TLR7 and TLR9 by the pDCs requires an intact endocytosis pathway. In addition, research supported during the first funding period of this R01 program demonstrated that pDCs utilize autophagy, a highly conserved pathway of delivering cytosolic materials to the endolysosomes, for recognition of cytosolic viral replication intermediates of ssRNA viruses. These results provided a critical link between TLR recognition of viruses and the type I IFN induction pathways, and suggested the importance of these and other TLRs in viral recognition and immune responses. However, major gaps in our understanding of the biology of pDCs relate to how the endosomal TLRs traffic within the cells to reach the appropriate compartment from which they can signal to induce type I IFNs and pro-inflammatory cytokines. In this proposal, we provide evidence in the Preliminary Studies section that demonstrate the role of adaptor protein 3 (AP-3) complex, phosphatidyl inositol 5 kinase, and Atg5 in intracellular trafficking and signaling by TLRs. Based on these data, we describe systematic approaches to examine the molecular and cellular mechanisms of innate recognition by TLR9 in pDCs. In the first Aim, we will determine the mechanism by which AP-3 complex mediates trafficking of endosomal TLRs in pDCs and define the cell biology of IFN- endosomes. In the second Aim, we propose to identify the phosphoinositide-mediated signaling mechanism in pDCs. In the final Aim, we will integrate all of our findings in the three separate pathways and interrogate a possible link between autophagy and TLR trafficking mediated by AP-3 and phosphoinositide-dependent pathways. Through basic understanding of the biology of pDCs, these studies will help to provide important foundation with which to design immunological interventions against viral diseases. PUBLIC HEALTH RELEVANCE: Plasmacytoid dendritic cells (pDCs) are critical innate immune cells that secrete high levels of type I interferons (IFNs), which play a pivotal role in the early innate immune responses against virus infection. In this application, we propose to examine the cell biological mechanism by which pDCs recognize viral pathogens through pattern recognition receptors, the Toll-like receptors. The understanding gained from the proposed studies will not only provide scientific advances in how pDCs detect viral pathogens, but also to help establish critical foundation with which to design immunological interventions and preventative measures against a wide variety of viral diseases.